Egg-laying Females Research Articles

Bacteria of the genus Erwinia found in the spermatheca of the laurel psyllid Trioza alacris.

Psylloidea are economically important insects causing serious damage to plants by direct feeding and/or vectoring bacterial pathogens. Results reported here indicate the presence of extracellular bacteria in the spermatheca of egg-laying Trioza alacris females. One phylotype, sharing 99% identity with the non-phytopathogenic bacterium Erwinia tasmaniensis, was identified regardless of methods applied or insect sampling year and location. This is the first study, achieved by ultrastructural, cultural, and 16S rRNA gene-based analysis, of an insect spermatheca microbiota.

Parental exposure to elevated pCO2 influences the reproductive success of copepods.

Substantial variations are reported for egg production and hatching rates of copepods exposed to elevated carbon dioxide concentrations (pCO2). One possible explanation, as found in other marine taxa, is that prior parental exposure to elevated pCO2 (and/or decreased pH) affects reproductive performance. Previous studies have adopted two distinct approaches, either (1) expose male and female copepoda to the test pCO2/pH scenarios, or (2) solely expose egg-laying females to the tests. Although the former approach is more realistic, the majority of studies have used the latter approach. Here, we investigated the variation in egg production and hatching success of Acartia tonsa between these two experimental designs, across five different pCO2 concentrations (385–6000 µatm pCO2). In addition, to determine the effect of pCO2 on the hatching success with no prior parental exposure, eggs produced and fertilized under ambient conditions were also exposed to these pCO2 scenarios. Significant variations were found between experimental designs, with approach (1) resulting in higher impacts; here >20% difference was seen in hatching success between experiments at 1000 µatm pCO2 scenarios (2100 year scenario), and >85% at 6000 µatm pCO2. This study highlights the potential to misrepresent the reproductive response of a species to elevated pCO2 dependent on parental exposure.

Habitat use and population biology of the Danube Clouded Yellow butterfly Colias myrmidone (Lepidoptera: Pieridae) in Romania

The Danube Clouded Yellow (Colias myrmidone) is one of the most endangered butterflies in Europe. Its dis- tribution range shrank dramatically in the last few decades due to the extinction of populations in Western and Central Eur- ope. Ecological studies were commenced when populations were already at the verge of extinction, thus our knowledge on the population ecology and habitat use of this butterfly is very limited. Here we report the results of a study on habitat pref- erences, egg distribution and demography of C. myrmidone in Romania, perhaps the last remaining stronghold of the species in Central Europe. We found that Danube Clouded Yellow adults occurred mainly in mesophilous grasslands created by forest clearing and then maintained by low intensity grazing allowing bushes and forest-edge vegetation to develop and host Chamaecytisus species to grow. Butterflies highly pre- ferred lightly grazed pastures over hay meadows and abandoned grasslands, their density was positively related to host plant density. Egg-laying females preferred habitat pat- ches with relatively high cover of the host plant and tall veg- etation. Both apparent survival rate and encounter probability were lower for females than males, and parameter estimations also had much higher errors for females. These indicate that much higher sampling effort is needed to estimate and monitor population parameters for females than for males. Our results provide guidelines for the habitat management and population monitoring of Colias myrmidone, thus may significantly contribute to its successful conservation.

Alternating Temperatures Affect the Performance ofTrichogrammaSpecies

The environmental compatibility of a biological control agent is an important aspect of successful reduction of agricultural pests. Temperature fluctuations during the day have a strong influence on the performance of laboratory-reared parasitoids. In field conditions, Trichogramma (Hymenoptera: Trichogrammatidae) wasps are exposed to variable temperatures during their development, which has a significant impact on their ability to control pest species. A simulation-based study was undertaken to evaluate the impact of variations in daily temperature on the pest-control abilities of female Trichogramma and their immature progenies. Considering the temperature variability of different agricultural zones of India, five temperature levels ranging from 20°C to 40°C were selected for daily short-term heat shocks to the immature progenies and egglaying females of two major Trichogramma species. Intensity and frequency of thermal shocks showed inverse relationships with adult emergence, fecundity, and longevity of T. chilonis and T. poliae. Parasitoid pupae were found to be more tolerant to temperature variations than eggs and larvae. Fecundity and longevity of parasitoids were significantly reduced under high temperature shocks to egg-laying females. Sex ratio was significantly affected by high temperature shocks to the immature and adult stages. However, the effect was more severe in eggs. A female-biased sex ratio was apparent in both parasitoids throughout the experiment. Overall, daily short-term temperature shocks to different developmental stages of parasitoids showed radical effects on emergence, fecundity, longevity, and sex ratio of the progeny. Therefore, releases of parasitoids should be conducted when they are in their pupal stages during the morning and evening in order to achieve their highest effectiveness for pest management.

Alternating temperatures affect the performance of Trichogramma species.

The environmental compatibility of a biological control agent is an important aspect of successful reduction of agricultural pests. Temperature fluctuations during the day have a strong influence on the performance of laboratory-reared parasitoids. In field conditions, Trichogramma (Hymenoptera: Trichogrammatidae) wasps are exposed to variable temperatures during their development, which has a significant impact on their ability to control pest species. A simulation-based study was undertaken to evaluate the impact of variations in daily temperature on the pest-control abilities of female Trichogramma and their immature progenies. Considering the temperature variability of different agricultural zones of India, five temperature levels ranging from 20ºC to 40ºC were selected for daily short-term heat shocks to the immature progenies and egg-laying females of two major Trichogramma species. Intensity and frequency of thermal shocks showed inverse relationships with adult emergence, fecundity, and longevity of T. chilonis and T. poliae . Parasitoid pupae were found to be more tolerant to temperature variations than eggs and larvae. Fecundity and longevity of parasitoids were significantly reduced under high temperature shocks to egg-laying females. Sex ratio was significantly affected by high temperature shocks to the immature and adult stages. However, the effect was more severe in eggs. A female-biased sex ratio was apparent in both parasitoids throughout the experiment. Overall, daily short-term temperature shocks to different developmental stages of parasitoids showed radical effects on emergence, fecundity, longevity, and sex ratio of the progeny. Therefore, releases of parasitoids should be conducted when they are in their pupal stages during the morning and evening in order to achieve their highest effectiveness for pest management.

First Report of the Root-Knot Nematode Meloidogyne ethiopica on Tomato and Cucumber in Turkey.

The root-knot nematode Meloidogyne ethiopica Whitehead, mainly reported from African countries, was first described in 1968 in Tanzania (4). It was further detected in South America (Brazil, Chile, and Peru) (2). In 2004, M. ethiopica was recorded for the first time in Europe on tomato (3) and later in field soil samples from maize (Zea mays L.) and kiwi [Actinidia deliciosa (A. Chev.) C. F. Liang & A. R. Ferguson] collected in the area of Kavalla in North Greece (1). M. ethiopica was included on the EPPO alert list of harmful organisms in 2011. In summer 2009, severe stunting, leaf wilting, and extensive root galling of a presumed root-knot nematode (Meloidogyne sp.) were detected during a routine survey for root nematodes attacking tomato (Solanum lycopersicum L.) crops from two greenhouses in the campus of University of Ondokuz Mayis and attacking cucumber (Cucumis sativus L.) crops from commercial greenhouses in Çarsamba district of Samsun Province, Turkey. Perineal patterns of Meloidogyne sp. females collected from infested roots were variable, with moderately high to high dorsal arch, lateral line mostly indistinct and striae usually wavy, which is very similar to that seen in M. incognita Kofoid & White. Such variability among perineal patterns precluded its accurate identification. For further species identification, protein extracted from young egg-laying females were resolved in 3% stacking (pH 6.8) and 7% separating (pH 8.8) polyacrylamide gel with Tris-glycine buffer. The gels were stained with α-naphthyl acetate substrate for esterase activity (1). The esterase phenotypes exhibited a three banded pattern, E3, that was identical to M. ethiopica. Therefore, esterase studies were repeated including nematodes from M. ethiopica culture from Slovenia used as a reference. In addition, small subunit rDNA sequence analyses were performed to complete species identification. DNA sequences of a 1.6-kb rDNA fragment were generated using two sets of primers (1) and deposited in the NCBI GenBank with the accession number KC551945. The sequence was compared to the corresponding fragments of M. ethiopica and closely related species from the NCBI GenBank. DNA sequence of M. ethiopica from Turkey exhibited the highest identity of 99.8% to the sequence FJ559408 of M. ethiopica from Slovenia. Sequences KC551945, FJ559408, AY942630, and JQ768373 of M. ethiopica from Turkey, Slovenia, Brazil, and Greece, respectively, clustered together and formed a separate clade using phylogenetic analyses. This species may pose a threat for vegetable production in Turkey since it has a wide host range infesting numerous higher plants including monocotyledons, dicotyledons, herbaceous, and woody plants. Furthermore, M. ethiopica may have been present in Turkey for many years because correct identification based only on perineal pattern is difficult. Thus, misidentification might have been possible. The distribution of this nematode in more extensive vegetable fields should be determined. To our knowledge, this is the first report of M. ethiopica in Turkey and the third finding of this species in Europe.

First Report of the Root-Knot Nematode Meloidogyne artiellia in Belgium

In 2011, second-stage juveniles (J2) of an unknown root-knot nematode (Meloidogyne spp.) were detected during a routine survey for root-knot nematodes on arable land in Harveng, Belgium, after a crop of wheat. Most of the loamy soil samples (36 out of 42) contained J2 of the common root-knot nematode M. naasi Franklin, 1965 (1), while 15 of these also contained the unknown species, albeit in lower densities (22 J2/100 ml vs. 157 J2/100 ml soil). After detailed morphological observation of the unknown J2, they were until further notice identified as Meloidogyne artiellia Franklin, 1961 (2), the British root-knot nematode. To confirm the identification, a pure culture of M. artiellia was established by adding nematode suspensions to pots planted with kale (Brassica oleracea var. laciniata), a non-host for M. naasi (3). After 2 months, Meloidogyne spp. females, males, and J2 were isolated from galled kale roots. Morphological characteristics (n = 25) from the perineal pattern (rounded with fine striae, lateral area with coarse ridges, angular dorsal arch) and stylet knobs (small, ovoid, and backwardly sloping) for the females, the head shape (set off with distinct head cap) and stylet knobs (small, ovoid and backwardly sloping) for the males, the hemizonid position (anterior, adjacent to S to E pore), tail shape (conical), and short tail length (18 to 27 μm) for the J2, fit with previous observed populations of M. artiellia (3). Young egg-laying females were used for isozyme electrophoresis, and showed typical malate dehydrogenase (N1b) and esterase (M2-VF1) patterns (3). Additionally, DNA was extracted from single juveniles by incubating them in a lysis buffer (200 mM NaCl, 200 mM Tris-HCl (pH 8), 1% β-mercaptoethanol and 800 μg/ml Proteinase K) during 1.5 h at 65°C and 5 min at 99°C in a thermocycler. One microliter of crude DNA extract was used for PCR. ITS-rDNA sequencing (GenBank Accession Numbers JX393299 and JX393300) confirmed the identity, showing a 98 to 100% homology with other M. artiellia sequences (AY150368 and AF248478). To our knowledge, this is the first report of the root-knot nematode, M. artiellia, in Belgium. This nematode has been reported from the Mediterranean area, where it causes damage on chickpea and wheat (4), as well as from the U.K. Its finding in Harveng, close to the French border, suggests a more extensive geographical distribution.

Prerequisites for evolution: variation and selection in yellow autumn birch leaves

Prerequisites for evolution: variation and selection in yellow autumn birch leaves

Olive Ridley Mass Nesting Ecology and Egg Harvest at Ostional Beach, Costa Rica

The olive ridley sea turtle (Lepidochelys olivacea), a species listed by the International Union for Conservation of Nature as vulnerable, is characterized by its mass-nesting behavior, also known as arribada. For decades, this phenomenon has facilitated the utilization of this species by coastal communities, much of it illegally. At Ostional beach, Costa Rica, a community-based egg-harvest program operates legally to generate important income for the community while promoting the protection of the ridley nesting assemblage. However, to date, no analysis exists that indicates the long-term viability of the egg harvest program as a sound management tool. To address this void, we generated baseline abundance data of the major arribada events that occurred in the period 2006–2010, as well as egg harvest data, along with preliminary hatching success. Arribadas ranged between 3564 and 476,550 egg-laying females, which indicated a large variability in the magnitude of the mass nesting events. Estimated mean egg harvest was 4746.4 and ranged from 1527 to 8138 total clutches. In relation to the estimated number of clutches laid, the estimated mean of clutches harvested was 21.2%, ranging from 1.5% to 102.4%. Estimated monthly mean hatching rates ranged from 0.0% to 32.6%. It is not clear whether arribadas underwent a significant change in abundance during the study period, although the number of years covered is too short to establish a long-term trend. However, when compared with historical data, the population appears to have declined. Based on our data, we present various management recommendations aimed at increasing hatching rates.

Maternal corticosterone is transferred into the egg yolk

Hormones deposited in the avian egg are considered in many studies to influence or to adjust offspring phenotype to prevailing conditions in an adaptive way. Several studies demonstrated an effect of corticosterone, the main glucocorticoid in birds, injected into the egg on the developing chick, but the injection of steroids in the egg is far from mimicking the natural distribution of the hormone in the egg. Other studies applied a stressor or corticosterone to the mother. However it is still debated whether an increase of circulating corticosterone in the mother translates into higher concentrations of corticosterone in the egg. Therefore, we investigated in captive barn owls Tyto alba whether circulating corticosterone in egg-laying females elevated within a physiological range, resulted in the deposition of corticosterone in eggs. We found that an increase in circulating corticosterone in the mother within the naturally occurring range translated into elevated concentrations of corticosterone in the yolk of subsequently laid eggs, indicating a specific time frame and yolk layer of corticosterone deposition. We conclude that increasing maternal plasma corticosterone within a naturally occurring range is an efficient tool to increase corticosterone concentration in the egg and to manipulate conditions for the developing embryo.

Genotypic variation in yellow autumn leaf colours explains aphid load in silver birch

• It has been suggested that autumn-migrating insects drive the evolution of autumn leaf colours. However, evidence of genetic variation in autumn leaf colours in natural tree populations and the link between the genetic variation and herbivore abundances has been lacking. • Here, we measured the size of the whole aphid community and the development of green-yellow leaf colours in six replicate trees of 19 silver birch (Betula pendula) genotypes at the beginning, in the middle and at the end of autumn colouration. We also calculated the difference between green leaf and leaf litter nitrogen (N) and estimated the changes in phloem sap N loading. • Autumn leaf colouration had significant genetic variation. During the last survey, genotypes that expressed the strongest leaf reflectance 2-4 wk earlier had an abundance of egg-laying Euceraphis betulae females. Surprisingly, the aphid community size during the first surveys explained N loss by the litter of different birch genotypes. • Our results are the first evidence at the tree intrapopulation genotypic level that autumn-migrating pests have the potential to drive the evolution of autumn leaf colours. They also stress the importance of recognizing the role of late-season tree-insect interactions in the evolution of herbivory resistance.

Nonvolatile Chemical Cues Affect Host-Plant Ranking by Gravid Polygonia c-album Females

In a multiple-choice test, the preference of egg-laying Polygonia c-album (comma butterfly) females was studied for oviposition on plants bearing surrogate leaves treated with crude methanol extracts obtained from leaves of seven host-plant species: Humulus lupulus, Urtica dioica, Ulmus glabra, Salix caprea, Ribes nigrum, Corylus avellana, and Betula pubescens. The ranking order of surrogate leaves treated with host-plant extracts corresponded well to that reported on natural foliage, except R. nigrum. Thus, host-plant choice in P. c-album seems to be highly dependent on chemical cues. Moreover, after two subsequent fractionations using reversed-phase chromatography the nonvolatile chemical cues residing in the most polar water-soluble fractions evidently provided sufficient information for egg-laying females to discriminate and rank between the samples of more and less preferred plants, since the ranking in these assays was similar to that for natural foliage or whole methanol extracts, while the physical traits of the surrogate leaves remained uniform.

Comparison of migration, penetration, development and reproduction of Meloidogyne graminicola on susceptible and resistant rice genotypes

Migration, penetration, development and reproduction of the rice root-knot nematode, Meloidogyne graminicola, in the resistant African rice genotypes TOG5674, TOG5675, CG14 and RAM131, and in the susceptible Asian rice genotypes IR64 and UPLRi-5 were studied and compared. The number of second-stage juveniles (J2) that had migrated horizontally and vertically towards the rhizosphere at 48 h after inoculation was comparable in both resistant and susceptible rice genotypes. Penetration of J2 was significantly lower in the resistant rice genotypes compared with the susceptible rice genotypes at 3 and 7 days after inoculation (DAI). Nematode development in the resistant rice genotypes was slower than in the susceptible rice genotypes. Nematode reproduction was significantly lower in the resistant rice genotypes, which supported fewer eggs per g of roots and eggs per female. A significantly higher percentage of egg-laying females was found in the susceptible rice genotypes. Mature females that had developed in the resistant rice genotypes were significantly smaller than the ones in the susceptible rice genotypes. The resistant rice genotypes had significantly fewer galls than the susceptible rice genotypes.

Ecological and evolutionary aspects of photothermal regulation of diapause in trichogrammatidae

Facultative winter diapause in species of the genus Trichogramma Westw. (Hymenoptera, Trichogrammatidae) is regulated, like in the majority of other insects, by photoperiod and temperature. In trichograms, prepupae are diapausing, but sensitivity to length of the light day and to temperature as to the cue factors inducing diapause is characteristic of all stages of development from embryo to the egg-laying female. The action of the cue factors affects not only the current, but also the subsequent generations. Under natural conditions the photothermal regulation provides the timely induction of the diapause coordinated both with astronomical year time (the photoperiodic reaction) and with specificity of this season (the temperature reaction). In the course of special experiments in trichograms there are also revealed the "rudimentary" reactions that have lost their adaptive role. Results of these studies prove once more that specificity of photoperiodic and temperature reactions inducing diapause and their relative importance and confinement to certain sensitive stages are determined not only by peculiarities of ecology of different insect taxa, but also by the course of their previous evolution.

Effects of incubation delay on viability and microbial growth of Wood Duck (Aix sponsa) eggs

Egg viability in birds declines with increasing length of the delay in the start of incubation (i.e., incubation delay) and may be influenced by microbial infection and exposure of eggs to temperatures above physiological zero (>24°C). Onset of incubation during egg laying results in developmental asynchrony of embryos but may help to maintain viability of early-laid eggs. We manipulated incubation delay of Wood Duck (Aix sponsa) eggs and tested for effects of incubation delay and ambient temperature on egg viability and microbial infection. We also examined onset of incubation by egg-laying females. Hatching success declined slowly with increasing length of incubation delay and was very weakly affected by increasing exposure to temperatures >24°C. On average, females began incubation at night 7 days after nest initiation, but egg viability had declined only 8.6% (95% confidence interval: 4.3–15.2) by day 7. Microbial infection of eggshells was not related to length of incubation delay or mean daily temperature of the exposure period. Clutch size declined as the breeding season progressed, and nocturnal incubation began earlier in the laying sequence with declining clutch sizes. Laying females, regardless of clutch size, incubated for 3 nights before starting full incubation. We suggest that early onset of incubation may be more important in reducing incubation period and predation risk than in maintaining viability of early-laid eggs.

Parasitism cost of living in a high quality habitat in the bog fritillary butterfly

Habitat quality and the impact of natural enemies might profoundly affect metapopulation dynamics and viability. However, their relative impact has usually been considered independently. Here we address the question of how caterpillar habitat quality and parasitism prevalence interact to shape habitat selection in the bog fritillary butterfly Boloria eunomia, parasitized at the caterpillar stage by a specialist wasp, Cotesia euno- miae. We first classified habitat quality by relating caterpillar density to descriptors of different microhabitat types. Second, we investigated parasitism prevalence in those dif- ferent microhabitats. Our results show that caterpillars and parasitoids mapped onto the same microhabitats, mainly zones with high abundance of the host plant. Accordingly, we suggest that both egg-laying females and parasitoids use the same cues for habitat selec- tion. As a consequence, there should be a fitness cost for B. eunomia females to lay their eggs in places where parasitism prevalence is high. We indeed detected that B. eunomia females frequently laid eggs in habitat types that presented suboptimal microhabitat conditions for caterpillars. This suggests that the lower parasitism prevalence in these suboptimal habitat types counterbalances lower caterpillar survival, leading to an overall similar survival in optimal and suboptimal habitat types. Spreading eggs in different habitat types is thus expected to be a safe strategy to mitigate the adverse possible effects of environmental stochasticity and parasitism prevalence on offspring survival unequal among microhabitat types. From a conservation viewpoint, the preservation of habitat heterogeneity, including optimal but also suboptimal habitat areas, is crucial to ensure persistence of both the host and its parasitoid.

A new method for catching cavity-nesting birds during egg laying and incubation

The physiological condition of female birds during the egg-laying and incubation periods is of considerable interest and yet is relatively understudied in wild birds, primarily due to the difficulty of catching birds during this period without causing nest desertion. We therefore developed a box-net to capture cavity-nesting birds using sections of a mist-net placed around a metal cubic frame. We captured female Great Tits (Parus major) as they left nest boxes during the egg-laying and incubation periods and measured desertion rates. Using box-nets, we captured 108 of 119 (90%) females during egg laying and 10 of 12 (83%) during incubation. Our recapture rate over two consecutive days during incubation was 50% (5 of 10). Females not captured left nest boxes before we attempted to capture them, escaped through a hole in the mist-net, or remained in nest boxes for more than 2 h, after which we ended capture attempts. Overall, 22% of egg-laying females deserted, with desertion rates highest early in the egg-laying period. Desertion rates of females captured using box-nets did not differ from those of undisturbed females. One of 10 females captured in a box-net deserted during the incubation period. Box-nets are portable, can be set up and taken down quickly and easily, and could potentially be used with nest boxes or natural cavities at any height. Box-nets are easy to construct and adaptable for use with an array of cavity-nesting birds, and can be an important tool for studying female physiology during egg laying and incubation.

Sperm Utilization Patterns and Reproductive Success in Captive Loggerhead Turtles (Caretta caretta)

Abstract We investigated the effects of the order of sequential matings in captive female loggerhead turtles (Caretta caretta) on the paternity of their successive clutches. Paternity analysis revealed that only 3 of the 7 successive clutches were multiply sired, although all egg-laying females were observed to copulate with multiple males prior to egg-laying. In multiply sired, successive clutches of particular females, the relative paternal contributions of different males did not vary, although some potentially successful matings were observed during internesting intervals. Prior to nesting periods, males showed mating in response to the female reproductive cycle in which the cumulative durations of mountings observed by highly sensitive cameras peaked at 21–40 days before any individual female laid her first clutch. A regression line between cumulative duration of mountings and relative parental contributions for 21–40 days before laying the first clutch fit a predictive equation for sperm competition...

The behavior and natural history of Chartergellus, a little-known genus of neotropical social wasps (Vespidae Polistinae Epiponini)

This study reviews published data on the behavior and natural history of Chartergellus and presents the first observations on social interactions in this genus of tropical swarm-founding wasps. Observations of Chartergellus golfitensis in Costa Rica and C. punctatior in Colombia showed that queens perform a post-oviposition egg-guarding vigil, and a bending display like that characteristic of epiponine social wasps that lack consistent morphological differences between workers and queens and have caste determination in the adult stage. Young, old, and queen (egg-laying) females of C. golfitensis showed small differences that indicate color changes with age, and structural differences that could be due to seasonal or colony-cycle changes in developmental conditions, but do not rule out the possibility of pre-adult caste determination, a phenomenon that needs to be carefully distinguished from pre-adult caste bias. Sexual dimorphism and the behavior of males at the nest in C. golfitensis is described, as well as the aggressive and avoidance behavior of females toward males. Nest structure in both species is as described previously for Chartergellus species, but some anomalies and their possible evolutionary significance are discussed. Cell initiation by an egg-laying queen, a behavior never seen by workers, and by a young female with slightly developed ovaries, may be vestiges of ancestral solitary reproductive traits where developed ovaries are associated with cell construction.

Egg spreading in the ant-parasitic butterfly, Maculinea alcon: from individual behaviour to egg distribution pattern

Oviposition site selection can be viewed as an act of parental care; it is a life history trait with significant consequences for offspring development and success. Herbivorous insects may adopt different strategies for spreading their eggs (e. g. single oviposition events versus egg clustering). Host plant use results from the interplay of available cues and the responsiveness of the ovipositing female. We studied how egg-laying behaviour of individual females resulted in the egg distribution patterns observed at the population level in the ant-parasitic butterfly Maculinea alcon. Eggs are laid on marsh gentians, Gentiana pneumonanthe, but survival also depends on the presence of Myrmica host ants as young caterpillars leave the host plant and survive only after being adopted by ants. Several studies have focused on egg distribution patterns, but less so on the behavioural mechanisms behind the oviposition patterns. Our observational data show evidence for some level of egg clustering, as predicted by the obligate myrmecophilous lifestyle. There was no evidence of egg-laying avoidance when gentians already carried several eggs. Phenological time windows for oviposition on individual flower buds and gentians were much wider than reported before. Detailed tracking of egg-laying females in the field showed high rates of oviposition and suggest relatively high average levels of fecundity compared to fully herbivorous lycaenid butterflies. Results are discussed in relation to adaptive oviposition strategies of this highly specialist ant parasite of conservation concern. (C) 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.